The present invention relates generally to stable reagent compositions, and storage containers therefor, for use as blood diluents and for assaying blood cells and platelets in an aliquot of a whole blood sample. The invention further relates to the long-term storage of such reagent compositions in packaging designed to maintain MCV-assays of blood samples aeration-insensitive and to maintain reagent stability over time.
The analysis of blood samples using semi-automated and fully-automated hematology analyzer instruments requires the use of specialized reagents in which blood samples are mixed and diluted. Such reagents allow the practitioner to obtain accurate, sensitive and precise measurements of a variety of blood cell parameters and to analyze the different blood cell types in a blood sample. As a particular example, hematology analysis of blood cells, especially red blood cells, platelets and reticulocytes, on automated instruments frequently involves the use of reagents comprising components which have limited stability overtime in storage.
For the analysis of blood samples containing red and white blood cells and platelets, blood samples are routinely drawn into containers (e.g., glass tubes and the like) for subsequent use and/or storage. When a blood sample is to be analyzed or tested, the container carrying the blood sample is opened and the sample is, in turn, exposed to the air. As a rule, venous blood contains about 100 times more dissolved carbon dioxide (as bicarbonate) than is present in the air. This is because red blood cells carry metabolic waste carbon dioxide from tissues to the lungs. When the container housing a venous blood sample is opened, the blood sample begins to be aerated and the surface of the red blood cell begins to be exposed to air-saturated plasma. This results in a driving force for the cells, i.e., red blood cells, to unload carbon dioxide to the air. Carbon dioxide diffuses out of the red cell membrane causing a measurable decrease in intracellular bicarbonate, and therefore, a decrease in osmolality. The cell simultaneously loses water to maintain the original osmolality. As a result, the cell shrinks during aeration. This is known as a sample handling xe2x80x9caeration effectxe2x80x9d. (Dacie and Lewis, Practical Haematology, fifth edition, p.38, Churchill Livingstone, 1975). With repeated blood samplings in air, the red blood cell MCV decreases and the sample handling effect is evidenced such that about 2-5% of the sample""s red blood cell volume is lost in a fully aerated sample of venous blood.
Virtually all hematology methods using conventional reagents and analyzer instrumentation are susceptible to the problem of sample handling effects, in which aeration of a blood sample induces red cell shrinkage, known as aeration-induced mean cell volume (MCV) shrinkage. The extent of such MCV shrinkage can be as high as 5%. As a blood sample is sampled repeatedly for various assays and mixed, the headspace (i.e., the volume of air above the blood) in the sample storage tube increases. As the tube is opened, there is contact between air and the plasma surrounding the blood cells in the sample, which can have a detrimental effect on the blood sample, particularly after repeated uses. The severity of such a sample handling aeration effect increases if the sample is sampled repeatedly with frequent remixing and opening of the tube to the air.
In addition to the above-described sample handling effect, another serious problem related to blood sample analysis is the problem of storage effect, which is also detrimental to the integrity and quality of the cells in a stored blood sample. Indeed, the actual storage of whole blood samples in closed containers (e.g., tubes) at room temperature for 24 hours may cause the MCV to increase as much as 7%, due to the metabolism of both the red and white blood cells in the closed container of blood. The metabolism leads to the accumulation of carbon dioxide and other osmotically active products which tend to increase the internal osmolality of the cells, thereby producing swelling during storage.
More specifically, to provide a theoretical mechanism not intended to be in any way limiting, during the storage of whole blood, carbon dioxide (CO2), a major metabolic product, diffuses from the white blood cells into the plasma and then into the red blood cells. Once inside the red cells, carbon dioxide is enzymatically hydrated to carbonic acid, which then dissociates to an osmotically active bicarbonate ion and a buffered proton. The dissociation is forced by the difference between the intracellular pH of the cell (approximately pH 7.4) and the pKa1 of carbonic acid. As a result of these steps, cellular osmolality increases. Water therefore simultaneously enters the red cells to restore the original internal osmolality of approximately 290 milliOsmoles/kg, to match the extracellular osmolality, and the cells swell. Carbon dioxide, which is generated within red cells, produces a similar effect. Storage of whole blood for 24 hours at room temperature (i.e., approximately 25xc2x0 C.) causes an approximately 5-7 fL (femtoliter) increase in MCV, at least part of which is due to the described process.
Thus, a goal of the present inventors was to develop reagents, which are stable over time, for use in blood sample analysis. The reagents of the present invention can reverse, decrease, or eliminate the above-described sample handling and storage effects on blood cells, i.e., by reducing aeration-induced-shrinkage or swelling from storage at room temperature. Correlative to this goal is the added need for novel storage devices and materials which provide storage packaging that maintains the stability of the newly developed reagents. Long-term stability provides considerable economic advantages to those in the art, since such reagents can be used, stored and re-used for longer periods of time before new reagents need to be purchased and used. In addition, stability of the reagent components over time is required to provide assurance to the user that the results obtained after use of the reagents with cells in hematology analysis will remain accurate, sensitive and precise following repeated use and long-term reagent storage.
Prior to the present invention, bicarbonate-containing reagents, e.g., blood diluents, that partially or completely reversed the effects of sample handling, e.g., aeration-induced MCV shrinkage, were not described. The art also fails to describe flexible, collapsible CO2 barrier packaging for such bicarbonate-containing diagnostic reagents, particularly for the purposes of long-term storage of such reagents, for hematology cell analysis and measuring the properties of red blood cells and other blood cells in a whole blood sample.
To address shrinkage of MCV due to aeration of blood, Bryner et al., (1997, xe2x80x9cThe Spun Micro-Hematocrit and Mean Cell Volume are Affected by Changes in the Oxygenation State of Red Blood Cellsxe2x80x9d, Clin. Lab. Haem., 19:99-103), proposed fully oxygenating blood samples by treatment with an equilibrating gas mixture containing oxygen, carbon dioxide and nitrogen and the use of a layer of mineral oil in the tube as a barrier to the loss of both oxygen and carbon dioxide from the blood samples equilibrated with the gas mixtures. However, because both gases are more soluble in mineral oil than in water, mineral oil is a very poor barrier for these gases. Another drawback of the approach proposed by Bryner et al. is that their treatment of opened tubes of blood with the equilibrating gas mixture is both time consuming and a potential biohazard. The approach of Bryner et al. involves the bubbling of air through blood which generates aerosols when the bubbles break. If the blood sample should contain pathogenic agents or substances, such aerosoling could be harmful to the handler, or others nearby, due, for example, to the inhalation of such pathogenic agent or substances.
In general, diluents employed for red blood cell analysis (e.g., Bayer H*(trademark) RBC Diluent) typically contain a very low bicarbonate concentration because of the problem of equilibration with atmospheric carbon dioxide, CO2. As an example, U.S. Pat. No. 4,971,917 to Kuroda describes a reticulocyte reagent containing 1-300 mmol/L of bicarbonate and a dye to enhance the staining of reticulocytes. This patent fails to disclose or appreciate that because CO2 is highly volatile, even when the bicarbonate concentration is as low as 20 mmol/L at pH 7 to 8, loss of CO2 to the air can lead to a rapid rise in pH. In addition, typical plastic reagent containers (e.g., polypropylene and polyethylene) are highly permeable to CO2. Such reagents stored in such containers therefore have very limited stability. The Kuroda patent also does not disclose the development of novel reagents and special storage devices for such reagents that insure long reagent stability over time.
Other blood diluent reagents contain the fixative glutaraldehyde as well as a detergent, such as SDS. In such reagents, as described by Kim and Ornstein, 1983, Cytometry, 3:419-427, the lytic action of the SDS was nullified by the formation of crosslinks within the cell by the glutaraldehyde fixative. However, because glutaraldehyde-containing reagents are commonly unstable and glutaraldehyde is believed to be an environmental hazard due to its toxicity and carcinogenicity, the use of such reagents is perceived as disadvantageous.
In addition, glutaraldehyde is volatile and as a component of such reagent compositions is unstable over long periods of storage, (xe2x80x9cGlutaraldehyde polymers in aqueous solutionxe2x80x9d, Anal. Biochem., 201:94-98). Also, when blood samples are diluted into or mixed with reagents containing glutaraldehyde, or other fixatives, sample handling effects are xe2x80x9clocked inxe2x80x9d due to the formation of the chemical crosslinks. Hence, methods employing glutaraldehyde-containing reagents tend to lock in the size of the blood cells in the tube at sampling time. However, the clinical utility of the mean cell volume (MCV) measurement would be improved if the MCV parameter reported by the hematology analyzer were closer to the in vivo MCV value of a fresh blood sample.
Until the present invention, no reagent composition that served as a blood sample diluent was able to reverse sample handling effects or to reduce sample storage effects at room temperature. By the present invention, a reagent composition is provided, and a storage container is fashioned, for long-term stability of the reagent during storage. The new reagent composition, which is free of crosslinker or fixative (e.g., glutaraldehyde) and comprises bicarbonate, surfactant as sphering agent and, optionally, a metal halide salt, can totally reverse the shrinking of cells caused by aeration and partly reverse the swelling of cells indicative of the xe2x80x9cstorage effectxe2x80x9d by up to 50%.
Use of the new reagent as a blood diluent allows the cellular bicarbonate and carbon dioxide content to equilibrate with those of the reagent which is xe2x80x9cmatchedxe2x80x9d to venous plasma. In addition, the absence of a crosslinking agent or fixative in the reagent of the present invention allows the cell volume to be affected by the bicarbonate in the reagent. Further, the newly provided flexible, collapsible, multi-layer, carbon dioxide-barrier bag storage container for use with the reagent has sufficiently low permeability to CO2 to show exceptional pH and reagent stability over time; for example, over two to three years.
Thus, the present invention provides needed solutions to the universal and significant problem of typical sample instabilities during use and storage that are encountered and lamented by practitioners in the hematology art. Since the solution to the problem requires a reagent having a relatively high concentration of CO2, the barrier bag provides the required long-term stability for such a reagent, and the collapsibility prevents air influx into the bag as reagent moved.